Phosphodiesterase 10

ABSTRACT

The present invention provides novel human PDE10 polypeptides, polynucleotides encoding the polypeptides, expression constructs comprising the polynucleotides, host cells transformed with the expression constructs; methods for producing PDE10 polypeptides; antisense polynucleotides; and antibodies specifically immunoreactive with the PDE10 polypeptides. The invention further provides methods to identify binding partners of PDE 10, and more particularly, binding partners that modulate PDE10 enzyme activity.

This application claims priority of U.S. Provisional Application No.60/075,508, filed Feb. 23, 1998.

FIELD OF THE INVENTION

The present invention relates generally to a novel phosphodiesterase(PDE) designated PDE10. Depending on nomenclature used, PDE10 is alsoreferred to as PDE9.

BACKGROUND OF THE INVENTION

Phosphodiesterases (PDEs) hydrolyze 3′, 5′ cyclic nucleotides to theirrespective nucleoside 5′ monophosphates. The cyclic nucleotides cAMP andcGMP are synthesized by adenylyl and guanylyl cyclases, respectively,and serve as second messengers in a number of cellular signalingpathways. The duration and strength of the second messenger signal is afunction of the rate of synthesis and the rate of hydrolysis of thecyclic nucleotide.

Multiple families of PDEs have been identified. The nomenclature systemincludes first a number that indicates the PDE family. To date, ninefamilies (PDE1-9) are known which are classified by: (i) primarystructure; (ii) substrate preference; (iii) response to differentmodulators; (iv) sensitivity to specific inhibitors; and (v) modes ofregulation [Loughney and Ferguson, in Phosphodiesterase Inhibitors,Schudt, et al. (Eds.), Academic Press New York, N.Y. (1996) pp. 1-19].The number indicating the family is followed by a capital letter,indicating a distinct gene, and the capital letter followed by a secondnumber, indicating a specific splice variant or a specific transcriptthat utilizes a unique transcription initiation site.

The amino acid sequences of all mammalian PDEs identified to dateinclude a highly conserved region of approximately 270 amino acidslocated in the carboxy terminal half of the protein [Charbonneau, etal., Proc. Natl. Acad. Sci. (USA) 83:9308-931,2 (1986)]. The conserveddomain includes the catalytic site for cAMP and/or cGMP hydrolysis andtwo putative zinc binding sites as well as family specific determinants[Beavo, Physiol. Rev. 75:725-748 (1995); Francis, et al., J. Biol. Chem.269:22477-22480 (1994)]. The amino terminal regions of the various PDEsare highly variable and include other family specific determinants suchas: (i) calmodulin binding sites (PDE1); (ii) non-catalytic cyclic GMPbinding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4);(iv) hydrophobic membrane association sites (PDE3); and (v)phosphorylation sites for either the calmodulin-dependent kinase II(PDE1), the cAMP-dependent kinase (PDE1, PDE3, PDE4), or the cGMPdependent kinase (PDE5) [Beavo, Physiol. Rev. 75:725-748 (1995);Manganiello, et al., Arch. Biochem. Acta 322:1-13 (1995); Conti, et al.,Physiol. Rev. 75:723-748 (1995)].

Members of the PDE1 family are activated by calcium-calmodulin. Threegenes have been identified; PDE1A and PDE1B preferentially hydrolyzecGMP while PDE1C has been shown to exhibit a high affinity for both cAMPand cGMP. The PDE2 family is characterized as being specificallystimulated by cGMP [Loughney and Ferguson, supra]. Only one gene hasbeen identified, PDE2A, the enzyme product of which is specificallyinhibited by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Enzymes in thePDE3 family are specifically inhibited by cGMP. Two genes are known,PDE3A and PDE3B, both having high affinity for both cAMP and cGMP,although the V_(max) for cGMP hydrolysis is low enough that cGMPfunctions as a competitive inhibitor for cAMP hydrolysis. PDE3 enzymesare specifically inhibited by milrinone and enoximone [Loughney andFerguson, supra]. The PDE4 family effects cAMP hydrolysis and includesfour genes, PDE4A, PDE4B, PDE4C, and PDE4D, each having multiple splicevariants. Members of this family are specifically inhibited by theanti-depressant drug rolipram. Members of PDE5 family bind cGMP atnon-catalytic sites and preferentially hydrolyze cGMP. Only one gene,PDE5A, has been identified. The photoreceptor PDE6 enzymes specificallyhydrolyze cGMP [Loughney and Ferguson, supra]. Genes include PDE6A andPDE6B (the protein products of which dimerize and bind two copies of asmaller γ inhibitory subunit to form rod PDE), in addition to PDE6Cwhich associates with three smaller proteins to form cone PDE. The PDE7family effects cAMP hydrolysis but, in contrast to the PDE4 family, isnot inhibited by rolipram [Loughney and Ferguson, supra]. Only one gene,PDE7A, has been identified. The PDE8 family has been shown to hydrolyzeboth cAMP and cGMP and is insensitive to inhibitors specific for PDEs1-5. Depending on nomenclature used, PDE8 is also referred to as PDE10,but is distinct from PDE10 described herein. The PDE9 familypreferentially hydrolyzes cAMP and is not sensitive to inhibition byrolipram, a PDE4-specific inhibitor, or isobutyl methyl xanthine (IBMX),a non-specific PDE inhibitor. Depending on nomenclature used, PDE9 isalso referred to as PDE8, but is distinct from PDE8 mentioned above. Todate, two genes have been identified in the PDE9 family.

Specific and non-specific inhibitors of the various PDE protein familieshave been shown to be effective in treating disorders attributable, inpart, to aberrant levels of cAMP or cGMP. For example, the PDE4-specificinhibitor rolipram, mentioned above as an anti-depressant, inhibitslipopolysaccharide-induced expression of TNFα and has been effective intreating multiple sclerosis in an animal model. Other PDE4-specificinhibitors are being investigated for use as anti-inflammatorytherapeutics, and efficacy in attenuating the late asthmatic response toallergen challenge has been demonstrated [Harbinson, et al., Eur.Respir. J. 10:1008-1014 (1997)]. Inhibitors specific for the PDE3 familyhave been approved for treatment of congestive heart failure. PDE5inhibitors are currently being evaluated for treatment of penileerectile dysfunction [Boolell, et al., Int. J. Impotence Res. 8:47-50(1996)]. Non-specific inhibitors, such as theophylline andpentoxifylline, are currently used in the treatment of respiratory andvascular disorders, respectively.

Given the importance of cAMP and cGMP in intracellular second messengersignaling, there thus exists an ongoing need in the art to identifyadditional PDE species. Identification of heretofore unknown families ofPDEs, and genes and splice variants thereof, will provide additionalpharmacological approaches to treating conditions in which cyclicnucleotide pathways are aberrant, as well as conditions in whichmodulation of intracellular cAMP and/or cGMP levels in certain celltypes is desirable. Identification of family-specific andenzyme-specific inhibitors will permit development of therapeutic andprophylactic agents which act on desired cell types expressing PDEsand/or particular metabolic pathways regulated by cyclic nucleotidemonophosphate steady-state concentrations.

SUMMARY OF THE INVENTION

In brief, the prevent invention provides purified and isolated PDE10polypeptides. Preferred polypeptides comprise the amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQID NO: 20 and SEQ ID NO: 22.

The invention also provides polynucleotides encoding polypeptides of theinvention. A preferred polynucleotide comprises the sequence set forthin SEQ ID NO: 1. Polynucleotides of the invention includepolynucleotides encoding a human PDE10 polypeptide selected from thegroup consisting of: a) the polynucleotide according to SEQ ID NO: 1,18, 20 or 22; b) a DNA which hybridizes under moderately stringentconditions to the non-coding strand of the polynucleotide of (a); and c)a DNA which would hybridize to the non-coding strand of thepolynucleotide of (a) but for the redundancy of the genetic code.Polynucleotides of the invention comprise any one of the polynucleotideset out in SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, as well asfragments thereof. The invention provide polynucleotides which are DNAmolecules. DNA molecules include cDNA, genomic DNA, and wholly orpartially chemically synthesized DNA molecule. The invention alsoprovides antisense polynucleotides which specifically hybridizes withthe complement of a polynucleotide of the invention.

The invention also provides expression constructs comprising apolynucleotide of the invention, host cells transformed or transfectedwith an expression construct of the invention, and methods for producinga PDE10 polypeptide comprising the steps of: a) growing the host cell ofthe invention under conditions appropriate for expression of the PDE10polypeptide and b) isolating the PDE10 polypeptide from the host cell orthe medium of its growth.

The invention further provides antibodies specifically immunoreactivewith a polypeptide of the invention. Preferably, the antibody is amonoclonal antibody. The invention also provides hybridomas whichproduces an antibody of the invention. Anti-idiotype antibodyspecifically immunoreactive with the antibody of the invention are alsocontemplated.

The invention also provides methods to identify a specific bindingpartner compound of a PDE10 polypeptide comprising the steps of: a)contacting the PDE10 polypeptide with a compound under conditions whichpermit binding between the compound and the PDE10 polypeptide; b)detecting binding of the compound to the PDE10 polypeptide; and c)identifying the compound as a specific binding partner of the PDE10polypeptide. Preferably, methods of the invention identify specificbinding partners that modulate activity of the PDE10 polypeptide. In oneaspect, the methods identify compounds that inhibits activity of thePDE10 polypeptide. In another aspect, the methods identify compoundsthat enhance activity of the PDE10 polypeptide.

The invention also provides methods to identify a specific bindingpartner compound of the PDE10 polynucleotide of the invention comprisingthe steps of: a) contacting the PDE1.0 polynucleotide with a compoundunder conditions which permit binding between the compound and the PDE10polynucleotide; b) detecting binding of the compound to the PDE10polynucleotide; and c) identifying the compound as a specific bindingpartner of the PDE10 polynucleotide. Preferably, the methods identifyspecific binding partner compounds that modulate expression of a PDE10polypeptide encoded by the PDE10 polynucleotide. In one aspect, methodof the invention identify compounds that inhibit expression of the PDE10polypeptide. In another aspect, methods of the invention identifycompounds that enhance expression of the PDE10 polypeptide.

Binding partner compounds identified by methods of the invention arealso contemplated, as are compositions comprising the compound. Theinvention further comprehends use of binding partner compounds of theinvention in production of medicaments for the treatment ofPDE10-related disorders.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptides and underlyingpolynucleotides for a novel PDE family designated PDE10. The PDE10family is distinguished from previously known PDE families in that itshows a lower degree of sequence homology than would be expected for amember of a known family of PDEs and it is not sensitive to inhibitorsthat are known to be specific for previously identified PDE families.Outside of the catalytic region of the protein, PDE10 shows littlehomology to other known PDEs. Even over the catalytic region, PDE10amino acid sequence identity is less than 40% when compared with thesame region in known PDEs. The invention includes both naturallyoccurring and non-naturally occurring PDE10 polynucleotides andpolypeptide products thereof. Naturally occurring PDE10 products includedistinct gene and, polypeptide species within the PDE10 family; thesespecies include those which are expressed within cells of the sameanimal as well as corresponding species homologs expressed in cells ofother animals. Within each PDE10 species, the invention further providessplice variants encoded by the same polynucleotide but which arise fromdistinct mRNA transcripts. Non-naturally occurring PDE10 productsinclude variants of the naturally occurring products such as analogs(i.e., wherein one or more amino acids are added, substituted, ordeleted) and those PDE10 products which include covalent modifications(i.e., fusion proteins, glycosylation variants, and the like).

The present invention provides novel purified and isolatedpolynucleotides (e.g., DNA sequences and RNA transcripts, both sense andcomplementary antisense strands, including splice variants thereof)encoding human PDE10s. DNA sequences of the invention include genomicand cDNA sequences as well as wholly or partially chemically synthesizedDNA sequences. Genomic DNA of the invention comprises the protein codingregion for a polypeptide of the invention and includes allelic variantsof the preferred polynucleotide of the invention. Genomic DNA of theinvention is distinguishable from genomic DNAs encoding polypeptidesother than PDE10 in that it includes the PDE10 coding region as definedby PDE10 cDNA of the invention. The invention therefore providesstructural, physical, and functional characterization for genomic PDE10DNA. Allelic variants are known in the art to be modified forms of awild type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild type genes, areinherently naturally occurring sequences (as opposed to non-naturallyoccurring variants which arise from in vitro manipulation).“Synthesized,” as used herein and is understood in the art, refers topurely chemical, as opposed to enzymatic, methods for producingpolynucleotides. “Wholly” synthesized DNA sequences are thereforeproduced entirely by chemical means, and “partially” synthesized DNAsembrace those wherein only portions of the resulting DNA were producedby chemical means. A preferred DNA sequence encoding a human PDE10polypeptide is set out in SEQ ID NO: 1. The worker of skill in the artwill readily appreciate that the preferred DNA of the inventioncomprises a double stranded molecule, for example the molecule havingthe sequence set forth in SEQ ID NO: 1 along with the complementarymolecule (the “non-coding strand” or “complement”) having a sequencededucible from the sequence of SEQ ID NO: 1 according to Watson-Crickbase paring rules for DNA. Also preferred are polynucleotides encodingthe PDE10 polypeptide of SEQ ID NO: 2.

The disclosure of a full length polynucleotide encoding a PDE10polypeptide makes readily available to the worker of ordinary skill inthe art every possible fragment of the full length polynucleotide. Theinvention therefore provides fragments of PDE10-encoding polynucleotidesof the invention comprising at least 10 to 20, and preferably at least15, nucleotides, however, the invention comprehends fragments of variouslengths. Preferably, fragment polynucleotides of the invention comprisesequences unique to the PDE10-encoding polynucleotide sequence, andtherefore hybridize under stringent or preferably moderate conditionsonly (i.e., “specifically”) to polynucleotides encoding PDE10, or PDE10polynucleotide fragments containing the unique sequence. Polynucleotidefragments of genomic sequences of the invention comprise not onlysequences unique to the coding region, but also include fragments of thefull length sequence derived from introns, regulatory regions, and/orother non-translated sequences. Sequences unique to polynucleotides ofthe invention are recognizable through sequence comparison to otherknown polynucleotides, and can be identified through use of alignmentprograms made available in public sequence databases.

The invention also provides fragment polynucleotides that are conservedin one or more polynucleotides encoding members of the PDE10 family ofpolypeptides. Such fragments include sequences characteristic of thefamily of PDE10 polynucleotides, and are also referred to as “signaturesequences.” The conserved signature sequences are readily discernablefollowing simple sequence comparison of polynucleotides encoding membersof the PDE10 family. Fragments of the invention can be labeled in amanner that permits their detection, and radioactive and non-radioactivelabeling are comprehended. Fragment polynucleotides are particularlyuseful as probes for detection of full length or other fragment PDE10polynucleotides. One or more fragment polynucleotides can be included inkits that are used to detect the presence of a polynucleotide encodingPDE10, or used to detect variations in a polynucleotide sequenceencoding PDE10.

The invention further embraces species homologs, preferably mammalian,of the human PDE10 DNA. The polynucleotide sequence information providedby the invention makes possible the identification and isolation ofpolynucleotides encoding related mammalian PDE10 molecules by well knowntechniques including Southern and/or Northern hybridization, andpolymerase chain reaction (PCR). Examples of related polynucleotidesinclude human and non-human genomic sequences, including allelicvariants, as well as polynucleotides encoding polypeptides homologous toPDE10 and structurally related polypeptides sharing one or morebiological, immunological, and/or physical properties of PDE10.

The invention also embraces DNA sequences encoding PDE10 species whichhybridize under moderately stringent conditions to the non-codingstrands, or complements, of the polynucleotide in any one of SEQ ID NOs:1, 18, 20, and 22. DNA sequences encoding PDE10 polypeptides which wouldhybridize thereto but for the redundancy of the genetic code arecontemplated by the invention. Exemplary moderate hybridizationconditions are as follows: hybridization at 65° C. in 3×SSC, 0.1%Sarkosyl, and 20 mM sodium phosphate, pH 6.8, and washing at 65° C. in2×SSC with 0.1% SDS. Exemplary high stringency conditions would includea final wash in 0.2×SSC/0.1% SDS, at 65° C. to 75° C. It is understoodin the art that conditions of equivalent stringency can be achievedthrough variation of temperature and buffer, or salt concentration asdescribed Ausebel, et al. (Eds.), Protocols in Molecular Biology, JohnWiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridizationconditions can be empirically determined or precisely calculated basedon the length and the percentage of guanosine/cytosine (GC) base pairingof the probe. The hybridization conditions can be calculated asdescribed in Sambrook, et al., (Eds.), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.(1989), pp. 9.47 to 9.51.

Autonomously replicating recombinant expression constructions such asplasmid and viral DNA vectors incorporating PDE10 sequences are alsoprovided. Expression constructs wherein PDE10-encoding polynucleotidesare operatively-linked to an endogenous or exogenous expression controlDNA sequence and a transcription terminator are also provided.

According to another aspect of the invention, host cells are provided,including procaryotic and eucaryotic cells, either stably or transientlytransformed with DNA sequences of the invention in a manner whichpermits expression of PDE10 polypeptides of the invention. Expressionsystems of the invention include bacterial, yeast, fungal, viral,invertebrate, and mammalian cells systems. Host cells of the inventionare a valuable source of immunogen for development of antibodiesspecifically immunoreactive with PDE10. Host cells of the invention arealso conspicuously useful in methods for large scale production of PDE10polypeptides wherein the cells are grown in a suitable culture mediumand the desired polypeptide products are isolated from the cells or fromthe medium in which the cells are grown by, for example, immunoaffinitypurification.

Knowledge of PDE10 DNA sequences allows for modification of cells topermit, or increase, expression of endogenous PDE10. Cells can bemodified (e.g., by homologous recombination) to provide increased PDE10expression by replacing, in whole or in part, the naturally occurringPDE10 promoter with all or part of a heterologous promoter so that thecells express PDE10 at higher levels. The heterologous promoter isinserted in such a manner that it is operatively-linked to PDE10encoding sequences. See, for example, PCT International Publication No.WO 94/12650, PCT International Publication No. WO 92/20808, and PCTInternational Publication No. WO 91/09955. The invention alsocomprehends that, in addition to heterologous promoter DNA, amplifiablemarker DNA (e.g., ada, dhfr, and the multifunctional CAD gene whichencodes carbamyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the PDE10 coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the PDE10 coding sequences in the cells.

The DNA sequence information provided by the present invention alsomakes possible the development through, e.g. homologous recombination or“knock-out” strategies [Capecchi, Science 244:1288-1292 (1989)], ofanimals that fail to express functional PDE10 or that express a variantof PDE10. Such animals are useful as models for studying the in vivoactivities of PDE10 and modulators of PDE10.

The invention also provides purified and isolated mammalian PDE10polypeptides as set out in SEQ ID NOs: 2, 19, 21, and 23. Presentlypreferred is a PDE10 polypeptide comprising the amino acid sequence setout in SEQ ID NO: 2. The invention embraces PDE10 polypeptides encodedby a DNA selected from the group consisting of: a) the DNA sequence setout in SEQ ID NOs:1, 18, 20, or 22; b) a DNA molecule which hybridizesunder stringent conditions to the noncoding strand of the protein codingportion of (a); and c) a DNA molecule that would hybridize to the DNA of(a) but for the degeneracy of the genetic code. The invention alsoembraces polypeptide fragments of the sequences set out in SEQ ID NOs:2, 19, 21, or 23 wherein the fragments maintain biological orimmunological properties of a PDE10 polypeptide. Preferred polypeptidefragments display antigenic properties unique to or specific for thePDE10 family of polypeptides. Fragments of the invention can be preparedby any the methods well known and routinely practiced in the art, havingthe desired biological and immunological properties.

The invention embraces polypeptides have at least 99%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55% and at least 50% identity and/orhomology to the preferred PDE10 polypeptide on the invention. Percentamino acid sequence “identity” with respect to the preferred polypeptideof the invention is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesin the PDE10 sequence after aligning both sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Percent sequence “homology” with respect to thepreferred polypeptide of the invention is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the PDE10 sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity. Conservativesubstitutions can be defined as set out below.

PDE10 polypeptides of the invention may be isolated from natural cellsources or may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofvarious host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. PDE10 products ofthe invention may be full length polypeptides, biologically orimmunologically active fragments, or variants thereof which retainspecific PDE10 biological or immunological activity. Variants maycomprise PDE10 polypeptide analogs wherein one or more of the specified(i.e., naturally encoded) amino acids is deleted or replaced or whereinone or more non-specified amino acids are added: (1) without loss of oneor more of the biological activities or immunological characteristicsspecific for PDE10; or (2) with specific disablement of a particularbiological activity of PDE10.

Variant products of the invention include mature PDE10 products i.e.,PDE10 products wherein leader or signal sequences are removed, andhaving additional, non-naturally occurring, amino terminal residues.PDE10 products having an additional methionine residue at position −1(Met⁻¹-PDE10) are contemplated, as are PDE10 products having additionalmethionine and lysine residues at positions −2 and −1(Met⁻²-Lys⁻¹-PDE10). Variants of these types are particularly useful,for recombinant protein production in bacterial cell types.

The invention also embraces PDE10 variants having additional amino acidresidues that result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide such as a glutathione-S-transferase (GST) fusion productprovide the desired polypeptide having an additional glycine residue atposition −1 as a result of cleavage of the GST component from thedesired polypeptide. Variants which result from expression in othervector systems are also contemplated.

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Conservative substitutions are recognizedin the art to classify amino acids according to their related physicalproperties and can be defined as set out in Table I (from WO 97/09433,page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996).TABLE I Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R Aromatic H F W Y Other N Q D E

Alternatively, conservative amino acids can be grouped as defined inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:N.Y.(1975), pp.71-77] as set out in Table II. TABLE II ConservativeSubstitutions II SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar(hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

The invention further embraces PDE10 products modified to include one ormore water soluble polymer attachments. Particularly preferred are PDE10products covalently modified with polyethylene glycol (PEG) subunits.Water soluble polymers may be bonded at specific positions, for exampleat the amino terminus of the PDE10 products, or randomly attached to oneor more side chains of the polypeptide.

Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, human antibodies CDR-grafted antibodies, or otherwise“humanized” antibodies, antigen binding antibody domains including Fab,Fab′, F(ab′)₂, F_(v), or single variable domains, and the like) andother binding proteins specific for PDE10 products or fragments,thereof. Specific binding proteins can be developed using isolated orrecombinant PDE10 products, PDE10 variants, or cells expressing suchproducts. The term “specific for” indicates that the variable regions ofthe antibodies recognize and bind PDE10 polypeptides exclusively (i.e.,able to distinguish PDE10 polypeptides from the superfamily of PDEpolypeptides despite sequence identity, homology, or similarity found inthe family of polypeptides), but may also interact with other proteins(for example, S. aureus protein A or other antibodies in ELISAtechniques) through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constant region ofthe molecule. Screening assays to determine binding specificity of anantibody of the invention are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(eds), Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognizeand bind fragments of the PDE10 polypeptides of the invention are alsocontemplated, provided that the antibodies are first and foremostspecific for, as defined above, PDE10 polypeptides. As with antibodiesthat are specific for full length PDE10 polypeptides, antibodies of theinvention that recognize PDE10 fragments are those which can distinguishPDE10 polypeptides from the superfamily of PDE polypeptides despiteinherent sequence identity, homology, or similarity found in the familyof proteins.

Binding proteins are useful for purifying PDE10 products and detectionor quantification of PDE10 products in fluid and tissue samples usingknown immunological procedures. Binding proteins are also manifestlyuseful in modulating (i.e., blocking, inhibiting or stimulating)biological activities of PDE10, especially those activities involved insignal transduction. Anti-idiotypic antibodies specific for anti-PDE10antibodies are also contemplated.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor PDE10 makes possible through use of Southern hybridization orpolymerase chain reaction (PCR) the identification of genomic DNAsequences encoding PDE10 and PDE10 expression control regulatorysequences such as promoters, operators, enhancers, repressors, and thelike. DNA/DNA hybridization procedures carried out with DNA sequences ofthe invention under moderately to highly stringent conditions arelikewise expected to allow the isolation of DNAs encoding allelicvariants of PDE10; allelic variants are known in the art to includestructurally related proteins sharing one or more of the biochemicaland/or immunological properties specific to PDE10. Similarly, non-humanspecies genes encoding proteins homologous to PDE10 can also beidentified by Southern and/or PCR analysis and useful in animal modelsfor PDE10-related disorders. As an alternative, complementation studiescan be useful for identifying other human PDE10 products as well asnon-human proteins, and DNAs encoding the proteins, sharing one or morebiological properties of PDE10. Polynucleotides of the invention arealso useful in hybridization assays to detect the capacity of cells toexpress PDE10. Polynucleotides of the invention may also be the basisfor diagnostic methods useful for identifying a genetic alteration(s) ina PDE10 locus that underlies a disease state or states.

The DNA and amino acid sequence information provided by the presentinvention also makes possible the systematic analysis of the structureand function of PDE10s. DNA and amino acid sequence information forPDE10 also permits identification of binding partner compounds withwhich a PDE10 polypeptide or polynucleotide will interact. Bindingpartner compounds include proteins and non-protein compounds such assmall molecules. Agents that modulate (i.e., increase, decrease, orblock) PDE10 activity or expression may be identified by incubating aputative, modulator with a PDE10 polypeptide or polynucleotide anddetermining the effect of the putative modulator on PDE10phosphodiesterase activity or expression. The selectivity of a compoundthat modulates the activity of the PDE10 can be evaluated by comparingits binding activity on the PDE10 to its activity on other PDE enzymes.Cell based methods, such as di-hybrid assays to identify DNAs encodingbinding compounds and split hybrid assays to identify inhibitors ofPDE10 polypeptide interaction with a known binding polypeptide, as wellas in vitro methods, including assays wherein a PDE10 polypeptide, PDE10polynucleotide, or a binding partner are immobilized, and solutionassays are contemplated under the invention.

Selective modulators may include, for example, antibodies and otherproteins or peptides which specifically bind to a PDE10 polypeptide or aPDE10-encoding nucleic acid, oligonucleotides which specifically bind toa PDE10, polypeptide or a PDE10 gene sequence, and other non-peptidecompounds (e.g., isolated or synthetic organic and inorganic molecules)which specifically react with a PDE10 polypeptide or underlying nucleicacid. Mutant PDE10 polypeptides which affect the enzymatic activity orcellular localization of the wild-type PDE10 polypeptides are alsocontemplated by the invention. Presently preferred targets for thedevelopment of selective modulators include, for example: (I) regions ofthe PDE10 polypeptide which contact other proteins and/or localize thePDE10 polypeptide within a cell, (2) regions of the PDE10 polypeptidewhich bind substrate, (3) cyclic nucleotide-binding site(s) of the PDE10polypeptide, (4) phosphorylation site(s) of the PDE10 polypeptide and(5) regions of the PDE10 polypeptide which are involved inmultimerization of PDE10 subunits. Still other selective modulatorsinclude those that recognize specific PDE10 encoding and regulatorypolynucleotide sequences. Modulators of PDE10 activity may betherapeutically useful in treatment of a wide range of diseases andphysiological conditions in which PDE activity is known to be involved.

PDE10 polypeptides of the invention are particularly amenable to use inhigh throughput screening assays to identify binding partners, andpreferably modulators. Cell based assays are contemplated, includingyeast based assay systems as well as mammalian cell expression systemsas described in Jayawickreme and Kost, Curr. Opin. Biotechnol.8:629-634(1997). Alternatively, automated and minaturized highthroughput screening (HTS) assays, such as high density free format highdensity screening, as described in Houston and Banks, Curr. Opin.Biotehcnol. 8:734-740 (1997). Combinatorial libraries are particularlyuseful in high throughput screening assays.

There are a number of different libraries used for the identification ofsmall molecule modulators, including, (1) chemical libraries, (2)natural product libraries, and (3) combinatorial libraries comprised ofrandom peptides, oligonucleotides or organic molecules.

Chemical libraries consist of structural analogs of known compounds orcompounds that are identified as “hits” or “leads” via natural productscreening. Natural product libraries are collections of microorganisms,animals, plants, or marine organisms which are used to create mixturesfor screening by: (1) fermentation and extraction of broths from soil,plant or marine microorganisms or (2) extraction of plants or marineorganisms. Natural product libraries include polyketides, non-ribosomalpeptides, and variants (non-naturally occurring) variants thereof. For areview, see Science 282:63-68 (1998). Combinatorial libraries arecomposed of large numbers of peptides, oligonucleotides, or organiccompounds as a mixture. They are relatively easy to prepare bytraditional automated synthesis methods, PCR, cloning or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include protein, peptidomimetic, multiparallel syntheticcollection, recombinatorial, and polypeptide libraries. For a review ofcombinatorial chemistry and libraries created therefrom, see Myers,Curr. Opion. Biotechnol. 8:701-707 (1997). Identification of modulatorsthrough use of the various libraries described herein permitsmodification of the candidate “hit” (or “lead”) to optimize the capacityof the “hit” to modulate activity.

Also made available by the invention are anti-sense polynucleotideswhich recognize and hybridize to polynucleotides encoding PDE10. Fulllength and fragment anti-sense polynucleotides are provided. The workerof ordinary skill will appreciate that fragment anti-sense molecules ofthe invention include (i) those which specifically recognize andhybridize to PDE10 RNA (as determined by sequence comparison of DNAencoding PDE10 to DNA encoding other known molecules) as well as (ii)those which recognize and hybridize to RNA encoding variants in thePDE10 family of proteins. Antisense polynucleotides that hybridize toRNA encoding other members of the PDE10 family of proteins are alsoidentifiable through sequence comparison to identify characteristic, orsignature, sequences for the family of molecules. Anti-sensepolynucleotides are particularly relevant to regulating expression ofPDE10 by those cells expressing PDE10 mRNA.

Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides)capable of specifically binding to PDE10 expression control sequences orPDE10 RNA are introduced into cells (e.g., by a viral vector orcolloidal dispersion system such as a liposome). The antisense nucleicacid binds to the PDE10 target nucleotide sequence in the cell andprevents transcription or translation of the target sequence.Phosphorothioate and methylphosphonate antisense oligonucleotides arespecifically contemplated for therapeutic use according to theinvention. The antisense oligonucleotides may be further modified bypoly-L-lysine, transferrin polylysine, or cholesterol moieties at the 5′end.

The invention further comprehends methods to modulate PDE10 expressionthrough use of ribozymes. For a review, see Gibson and Shillitoe, Mol.Biotech. 7:125-137 (1997). Ribozyme technology can be utilized toinhibit translation of PDE10 mRNA in a sequence specific manner through(i) the hybridization of a complementary RNA to a target mRNA and (ii)cleavage of the hybridized mRNA through nuclease activity inherent tothe complementary strand. Ribozymes can identified by empirical methodsbut more preferably are specifically designed based on accessible siteson the target mRNA [Bramlage, et al., Trends in Biotech 16:434-438(1998).] Delivery of ribozymes to target cells can be accomplished usingeither exogenous or endogenous delivery techniques well known androutinely practiced in the art. Exogenous delivery methods can includeuse of targeting liposomes or direct local injection. Endogenous methodsinclude use of viral vectors and non-viral plasmids.

Ribozymes can specifically modulate expression of PDE10 when designed tobe complementary to regions unique to a polynucleotide encoding PDE10.“Specifically modulate” therefore is intended to mean that ribozymes ofthe invention recognizes only a polynucleotide encoding PDE10.Similarly, ribozymes can be designed to modulate expression of all orsome of the PDE10 family of proteins. Ribozymes of this type aredesigned to recognize polynucleotide sequences conserved in all or someof the polynucleotides which encode the family of proteins.

The invention further embraces methods to modulate transcription ofPDE10 through use of oligonucleotide-directed triplet helix formation.For a review, see Lavrovsky, et al., Biochem. Mol. Med. 62:11-22 (1997).Triplet helix formation is accomplished using sequence specificoligonucleotides which hybridize to double stranded DNA in the majorgroove as defined in the Watson-Crick model. Hybridization of a sequencespecific oligonucleotide can thereafter modulate activity of DNA-bindingproteins, including, for example, transcription factors and polymerases.Preferred target sequences for hybridization include promoter andenhancer regions to permit transcriptional regulation of PDE10expression. Oligonucleotides which are capable of triplet helixformation are also useful for site-specific covalent modification oftarget DNA sequences. Oligonucleotides useful for covalent modificationare coupled to various DNA damaging agents as described in Lavrovsky, etal. [supra].

The invention comprehends mutations in the PDE10 gene that result inloss of normal function of the PDE10 gene product and underlie humandisease states in which failure of the PDE10 is involved. Gene therapyto restore PDE10 activity would thus be indicated in treating thosedisease states. Delivery of a functional PDE10 gene to appropriate cellsis effected ex vivo, in situ, or in vivo by use of vectors, and moreparticularly viral vectors (e.g., adenovirus, adeno-associated virus, ora retrovirus), or ex vivo by use of physical DNA transfer methods (e.g.,liposomes or chemical treatments). See, for example, Anderson, Nature,supplement to vol. 392, no. 6679, pp.25-20 (1998). For additionalreviews of gene therapy technology see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller,Nature, 357: 455-460 (1992). Alternatively, it is contemplated that inother human disease states, preventing the expression of or inhibitingthe activity of PDE10 will be useful in treating the disease states. Itis contemplated that antisense therapy or gene therapy could be appliedto negatively regulate the expression of PDE10.

Identification of modulators of PDE10 expression and/or biologicalactivity provides methods to treat disease states that arise fromaberrant PDE10 activity. Modulators may be prepared in compositions foradministration, and preferably include one or more pharmaceuticallyacceptable carriers, such as pharmaceutically acceptable (i.e., sterileand non-toxic) liquid, semisolid, or solid diluents that serve aspharmaceutical vehicles, excipients, or media. Any diluent known in theart may be used. Exemplary diluents include, but are not limited to,polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma. The modulator compositions canbe packaged in forms convenient for delivery. The compositions can beenclosed within a capsule, sachet, cachet, gelatin, paper, or othercontainer. These delivery forms are preferred when compatible with entryof the composition into the recipient organism and, particularly, whenthe composition is being delivered in unit dose form. The dosage unitscan be packaged, e.g., in tablets, capsules, suppositories or cachets.The compositions may be introduced into the subject by any, conventionalmethod including, e.g., by intravenous, intradermal, intramuscular,intramammary, intraperitoneal, or subcutaneous injection; by oral,sublingual, nasal, anal, vaginal, or transdermal delivery; or bysurgical implantation, e.g., embedded under the splenic capsule or inthe cornea. The treatment may consist of a single dose or a plurality ofdoses over a period of time.

The invention also embraces use of a PDE10 polypeptide, a PDE10polynucleotide, or a binding partner thereof in production of amedicament for treatment of a PDE10-related biological disorder.

The present invention is illustrated by the following examples relatingto the isolation of a polynucleotide encoding a PDE10 polypeptide andexpression thereof. Example 1 describes identification of an ESTencoding a partial PDE10 polypeptide and isolation of a full lengthPDE10-encoding clone. Example 2 relates to Northern blot analysis ofPDE10 expression. Example 3 addresses chromosome mapping of PDE10.Example 4 describes expression and characterization of a recombinantPDE10 polypeptide. Example 5 describes production of anti-PDE10antibodies. Example 6 provides an analysis of PDE10 expression using insitu hybridization. Example 7 relates to high throughput screening toidentify inhibitors of PDE10.

EXAMPLE 1, Identification of an EST Related to a Human PDE and Isolationof a Full Length PDE10-Encoding Polynucleotide

Using the sequences of known human, 3′, 5′ cyclic nucleotidephosphodiesterases, a search of the National Center for BiotechnologyInformation (NCBI) Expressed Sequence Tags (EST) database was undertakenin order to identify cDNA fragments that could potentially be useful forthe identification of novel phosphodiesterase (PDE) genes. This databasecontains DNA sequences representing one or both ends of cDNAs collectedfrom a variety of tissue sources. A single sequencing run is performedon one or both ends of the cDNA and the quality of the DNA sequencevaries tremendously. At the time the PDE searches were performed, theEST sequence database contained more than 600,000 cDNA sequences from avariety of organisms.

The search for novel PDE sequences included three steps. First, theBLASTN program available through NCBI was used to identify DNA sequencesin the EST sequence database with homology to cDNA sequences encodingknown human PDEs. The program compares a nucleotide query sequenceagainst a nucleotide sequence database. The cDNA sequences of thefifteen known human PDEs were submitted and fifteen BLASTN searches wereperformed; the query PDE sequences included PDE1A3 [Loughney, et al., J.Biol. Chem. 271:796-806 (1996)], PDE1B1 [Yu, et al., Cell Signaling,9:519-529 (1997)], PDE1C2 [Loughney, et al., J. Biol. Chem. 271:796-806(1996)], PDE2A3 [Rosman, et al., Gene 191:89-95 (1997)], PDE3A [Meacci,et al., Proc. Natl. Acad. Sci. (USA) 89:3721-3725 (1992)], PDE3B [Mikiet al., Genomics 36:476-485 (1996)], PDE4A5 [Bolger, et al., Mol. Cell.Biol. 13:6558-6571 (1993)], PDE4B2 [Bolger, et al., Mol. Cell. Biol.13:6558-6571 (1993)], PDE4C [Bolger, et al., Mol. Cell. Biol.13:6558-6571 (1993)], PDE4D1 [Bolger, et al., Biochem. J. 328:539-548(1997)] and PDE4D3 [Bolger, et al., Mol. Cell. Biol. 13:6558-6571(1993)], PDE5A, PDE6A [Pittler, et al., Genomics 6:272-283 (1990)],PDE6B [Collins, et al., Genomics 13:698-704 (1992)], PDE6C [Piriev, etal., Genomics 28:429-435 (1995), and PDE7A1 [Michaeli, et al., J. Biol.Chem. 17:12925-12932 (1993)]. The BLASTN results were examined and ESTsequences that were judged as corresponding to each of the fifteen knownPDE cDNAs were identified and collected into a table. The PDE6A andPDE6B sequences used as queries were truncated at 3′, end (removing aportion of the 3′ untranslated region) due to the presence of repetitiveelements in the 3′ untranslated region of the cDNAs.

Secondly, the NCBI TBLASTN program was used to examine the homologybetween the protein sequence of the fifteen known human PDEs (as above)and the six different possible proteins encoded by each of the EST DNAsequences. In this search, the EST sequences are translated in the sixpossible reading frames and the amino acid sequences generated arecompared to the query PDE amino acid sequences. Sequences identified ashomologous at the amino acid level were examined and any EST sequencespositively identified as corresponding to a known PDE during the BLASTNsearch described above were discarded.

The third step of the search involved analyzing the sequences that werenot known PDEs. These amino acid sequences were homologous to a knownPDE but were not identified as one of the 15 known PDE genes during theBLASTN searches.

The initial BLAST searches identified three EST sequences, designatedX88347 (SEQ ID NO: 3), X88467 (SEQ ID NO: 4), and X88465 (SEQ ID NO: 5),that were obtained from an exon trapping experiment using chromosome 21genomic DNA and found to encode an amino acid sequence having homologyto the catalytic region of one or more of the PDE query sequences.X88347 showed homology with the amino acid sequences of PDE1A, 1B, 1C,3A, 3B, 4A, 4B and 4D; X88467 showed homology to PDE1A, 1B, 1C, 4A, 4B,4C, and D4; and X88465 was homologous to PDE1A and 1B amino acidsequences. At the 5′ terminus, EST X88465 was 58 nucleotides shorterthan was X88467 and was not considered further.

When X88347 was translated from nucleotides 1-222 and the resultantprotein was compared to PDE1A, the two proteins were the same at 23 of51 amino acid positions (45% identity). When X88467 was translated fromnucleotide 3 to 155 and the resultant protein compared to PDE1A, 15 of36 amino acids were the same (42% identity). Because ESTs X88347 andX88467 showed homology to two different regions of the catalytic regionof PDE1A, it seemed possible that they represented two different exonsfrom a novel PDE gene.

X88347 was used as a query in a BLASTN search of the NCBI EST database.In addition to itself, X88347 identified three other human EST sequenceswith high enough homology to suggest the sequences were derived from thesame gene. EST R00718, (SEQ ID NO: 6) showed 91% identity to X88347.R00719 (SEQ ID NO: 7) represented the 3′-end of the same cDNA as R00718.R45187 (SEQ ID NO: 8) showed 88% identity to X88347. Two mouse cDNAswere also identified; W82786 (SEQ ID NO: 9) (91% identity) and W10517(SEQ ID NO: 10) appeared to represent the mouse homolog of X88347. ABLASTN search using W10517 as probe identified another sequence H90802(SEQ ID NO: 11), which appeared to represent another human EST that maybe part of the human PDE gene. The several human cDNAs were notidentical to each other, and the quality of the sequencing was poor. ThecDNA represented by the R00719 and R00718 EST sequences was obtainedfrom the American Type Culture Collection (Rockville, Md.) whichmaintains and makes publicly available deposits of ESTs identified andsequenced by I.M.A.G.E., Lawrence Livermore National Laboratory,(Livermore, Calif.). The cDNA had been isolated from a fetal liver andspleen library and mapped to chromosome 21.

R00718/9 was sequenced upon receipt and found to be consistent with theEST database sequence. The polynucleotide and amino acids sequences forR00718/9 are set out in SEQ ID Nos: 12 and 13, respectively. TheR00718/9 clone contained a 0.6 kb insert with a poly A tail at the3′-end. The open reading frame encoded a protein with homology to otherPDEs but did not extend to the 5′end of the cDNA. Beginning at aminoacid position 9, a QSDRE sequence was found. Corresponding D and Eresidues were found within all of the query sequences. Query sequencesalso included a conserved E(F/Y) sequence located amino terminal to theconserved D and E residues, but this sequence was not found in ESTR00718/9. Instead, the EST contained eight amino acids followed by astop codon. The R00718/9 cDNA appeared to diverge from the PDE querysequences in the catalytic region and the open reading frame was notmaintained. The disrupted open reading frame may suggest the presence ofan intron that had not been removed or that the R00718/9 sequence wasjoined to some unidentified extraneous polynucleotide sequence. The generepresented by R00718/9 was designated PDE10.

In order to identify additional PDE10 sequences, a probe was generatedbased on the PDE10 sequence and used to screen cDNA libraries. First,two primers, R71S100R (SEQ ID NO: 14) and R71A521H (SEQ ID NO: 15) weresynthesized for use in PCR to amplify a 420 nucleotide portion of theR00718/9 DNA fragment (nucleotides 130 to 550). Primer R71S100Rgenerated an EcoRI restriction site in the amplification product(underlined below) and primer R71A521H generated a HindIII site (alsounderlined below). The PCR fragment was designed to include the regionof R00718/9 homologous to other PDEs, but not the poly A tail. R71S100R(SEQ ID NO: 14) AGTCGAATTCACCGTGAGAAGTCAGAAG R71A521H (SEQ ID NO: 15)GTCAAAGCTTACATGGTCTTGTGGTGCCThe PCR reaction contained 50 pg R00719 cDNA, 10 ng/μl each primer, 0.2mM dNTP, 1×PCR buffer (Perkin-Elmer), 2 mM MgCl₂, and 1.25 U Taqpolymerase (Perkin-Elmer). The reaction was first maintained at 94° C.for four minutes, after which thirty cycles of one minute 94° C., twominutes 50° C., and four minutes at 72° C. were performed. The PCRfragment was purified using low melting point agarose gelelectrophoresis.

For library screening, the PCR fragment was labeled with ³²P with arandom priming kit (Boehringer Mannheim) according to manufacturer'sinstructions and used to screen 10⁶ cDNAs from a human heart cDNAlibrary (Stratagene, La Jolla, Calif.), 5×10 ⁵ cDNAs from a humanhippocampal cDNA library (Clontech, Palo Alto, Calif.), and 7.5×10⁵cDNAs from a human fetal brain cDNA library (Stratagene). Hybridizationwas carried out overnight in buffer containing 3×SSC, 0.1% Sarkosyl, 20mM sodium phosphate, pH 6.8, 10× Denhardt's solution, and 50 μg/mlsalmon sperm DNA at 65° C. Eleven positives were obtained from the fetalbrain library and three from the hippocampal library. Partial sequencingled to the selection of one, FB79c, for further characterization. Thepolynucleotide and deduced amino acid sequences for FB79c are set out inSEQ ID NOs: 16 and 17, respectively.

FB79c contained a 1.3 kb insert; the 3′end of FB79c extended furtherthan that of R00718/9 and contained 12 adenosine residues of the poly Atail of R00718/9, an EcoRI site (GGAATTC), an additional fifty-ninenucleotides and a poly A sequence. At the 5′end, the sequence for FB79cdiffered from that of R00718/9 beginning at, and continuing 5′ of,nucleotide 121 of R00718/9 (corresponding to nucleotide 744 of FB79c).The open reading frame in FB79c (encoding a protein with homology to thequery PDEs) did not extend to the 5′end of the cDNA but ended in a stopcodon at nucleotide 104.

A sequence within the FB79c DNA located upstream of the point ofdivergence from R00718/9 (but within the portion of the open readingframe with homology to the other PDEs) was the region chosen for a probein subsequent library screening. The isolated sequence selected was a0.36 kb EcoRI fragment extending from nucleotide 308 to nucleotide 671of FB79c and was used to screen 1.75×10⁶ cDNAs from the fetal brain cDNAlibrary (Stratagene). More than twenty cDNAs were identified and twelvewere subjected to partial restriction mapping and DNA sequencing. Moreextensive sequencing on six of them led to the selection of clonesFB76.2 and FB68.2 for complete sequencing. The polynucleotide and aminoacid sequences for clone FB76.2 are set out in SEQ ID NOs: 18 and 19,respectively, and the polynucleotide and amino acid sequences for cloneFB68.2 are set out in SEQ ID NOs. 20 and 21, respectively.

FB76.2 contained a 1.9 kb cDNA insert; the 3′end of the cDNA stopped onenucleotide short of the poly A tail found in clone FB79c and thesequence diverged from FB79c 5′ of nucleotide 109 in clone FB79c(corresponding to nucleotide 715 in FB76.2). The open reading frame inthe FB76.2 sequence that encoded a protein with homology to the PDEquery sequences extended to the 5′end of the cDNA and the firstmethionine was encoded beginning at nucleotide 74. Assuming this residueto be the initiating methionine, the open reading frame of FB76.2encoded a 533 amino acid protein with a predicted molecular weight of61,708 Da.

Clone FB68.2 contained a 2 kb cDNA insert. At the 3′end, it extended tothe poly A tail found in the FB79c sequence and the open reading frameextended to the 5′end of the cDNA. FB68.2 differed from FB76.2 by thepresence of an additional internal 180 nucleotides (nucleotides 225 to404 of FB68.2) following corresponding nucleotide 335 of FB76.2. Sincethe number of additional nucleotides in the FB68.2 insertion wasdivisible by three, it did not alter the reading frame as compared toFB76.2. The position of the insert with respect to maintaining the samereading frame suggested that the sequence might represent an exon foundin some, but not all, PDE10 cDNAs. Alternatively, the additionalsequence could be an intron that had not been removed from the FB68.2cDNA.

Because the FB76.2 and FB68.2 differed from each other, additional PDE10DNAs were obtained and analyzed to more accurately define the PDE10nucleotide sequence. A 5′ 0.3 kb EcoRI fragment of FB76.2 (correspondingto nucleotides 1 to 285) was isolated and used as a probe to screen7.5×10⁵ cDNAs from the fetal brain cDNA library. Thirty seven positiveswere obtained, of which nineteen were first characterized with respectto fragment size (insert) that hybridized to the 0.3 kb EcoRI probe.Eight of the nineteen clones were subsequently characterized by partialsequencing. Two clones, FB93a and FB94a, contained 0.5 kb and 1.6 kbEcoRI fragments, respectively, that hybridized and were chosen forcomplete sequencing. The polynucleotide and amino acid sequences forclone FB93a are set out in SEQ ID NOs: 22 and 23, respectively, and thepolynucleotide and amino acid sequences for clone FB94a are set out inSEQ ID NOs 1 and 2, respectively.

FB93a contained a 1.5 kb insert which did not extend to the 3′end ofFB76.2 but was ninety nucleotides longer than FB76.2 at the 5′end. Theadditional nucleotides encoded a stop codon beginning at position 47which was in reading frame with the first methionine in FB76.2 describedabove (nucleotide 164 in FB93a). The position of the stop codonindicated the presence of a complete open reading frame and that FB76.2probably represented a full length cDNA. Like FB76.2, FB93a did notcontain the 180 nucleotide insert that was present in FB68.2.

FB94a contained a 1.5 kb cDNA insert and the 3′end extended almost 0.1kb beyond the stop codon. The first methionine was encoded beginning atnucleotide 26, and assuming this residue to be the initiatingmethionine, FB94a encoded a 466 amino acid protein with a predictedmolecular weight of 54,367 Da. FB194a differed from FB76.2 and FB93a byabsence of a 149 nucleotide region which, if consistent with thesequences for FB76.2 and FB93a, would have been located after nucleotide42. The absence of the 149 nucleotide sequence produced a putativeinitiator methionine that is in a different reading frame than thatfound in FB76.2 and FB93a. Like FB76.2 and FB93a, FB94a did not containthe 180 nucleotide region found in FB68.2.

A search of the EST data base with the FB94a and FB93a sequencesidentified yet another possible sequence for a PDE10 cDNA. The sequenceof EST A158.300 lacked both the 149 nucleotide and the 180 nucleotidesequences discussed above. In addition, A158300 also lacked 55nucleotides immediately 3′ to the 180 nucleotide region as found in theFB68.2 sequence. The open reading frame in A158300 extended to the 5′endand the first methionine corresponded to the same one used by FB76.2 andFB93a. The presence of the additional 55 nucleotide deletion fromA158300 resulted in a different reading frame fir the sequence betweenthe site where the 149 nucleotides were deleted and the site where the180 nucleotides were deleted.

The sequence information for PDE10 derived from these cDNA sequences canbe summarized as follows. There is a 149 nucleotide sequence found insome clones. (sequences FB76.2, FB93a, FB68.2) but not in all (sequencesFB94a, A158300). The 149 nucleotide sequence is followed by a 44nucleotide region that is present in all the PDE10 cDNAs analyzed todate. Following the 44 nucleotide region is a sequence of 235nucleotides in length. The region can be present in its entirety (asfound in the sequence for FB68.2) or without the first 180 nucleotides(as observed in sequences FB76.2 and FB93a). As still anotheralternative, the whole region can be removed (as found in the sequencefor A158300). These possibilities predict six different mRNA structures,four of which have been isolated.

The presence or absence of the 149 nucleotide region may reflect thepresence or absence of an exon, and the presence of all or some of the235 nucleotide region may reflect alternative 3′splice acceptor siteusage. As an alternative, it is also possible that the 235 nucleotideregion represents two separate exons of 180 and 55 nucleotides inlength. The presence or absence of the 149 nucleotide sequence altersthe reading frame of the encoded protein as does the presence or absenceof the 55 nucleotide sequence.

A number of single nucleotide differences have been observed incomparison of the various PDE10 cDNAs. R00718/9 has a cytosine atnucleotide position 155 whereas the other cDNAs have a thymidine at thisposition; this difference represented a silent change as proline isencoded by both sequences. R00718/9 also has a cytosine at position 161whereas the other cDNAs have an adenosine at the same position; thisdifference also represented a silent change as both sequences encodealanine. FB94a has a guanosine at position 1383 whereas the other cDNAshave an adenosine at this position; as a consequence of the difference,FB94a encodes a glycine rather than a glutamic acid at that position.FB76.2 has an adenosine rather than a cytosine at position 1809; thedifference does not effect an amino acid difference since the nucleotideposition is located in the 3′ untranslated region. FB79c also has oneless adenosine in the string of nucleotides between 1204 and 1215 thando the other cDNAs; this difference is also within the 3′ untranslatedregion.

In comparison of a predicted PDE10 amino acid sequence with other knownPDEs indicated that most, but not all, of the amino acids that areconserved among the query sequences were also found in PDE10. Comparisonof the PDE10 catalytic region to PDE4A, PDE5A, and PDE7A revealed 32%,30% and 34% identity, respectively.

EXAMPLE 2 Northern Blot

In order to determine which cell and tissue types express PDE10,Northern blot analysis was carried out using a commercially preparedmulti-tissue Northern blot (Clontech, Palo Alto, Calif.). The probe wasa EcoRI/BclI fragment of the FB76.2 corresponding to nucleotides 0.1 to883. Hybridization conditions were as previously described [Loughney etal., supra, (1996)].

Results indicated a 2.2 to 2.4 kb band which was strongest in kidney,present in heart, pancreas, and placenta, and weakest in brain, lung,skeletal muscle and liver. The band was fairly wide in placentasuggesting that it might contain a number of mRNAs of slightly differentsizes.

EXAMPLE 3 Chromosome Mapping

As mentioned above, the X88347, X88467, and X88465 ESTs were identifiedwith an exon trapping procedure using DNA from chromosome 21 [Chen etal. 1996]. X88467 was identified as a new sequence with homology to amouse calcium-, calmodulin-dependent phosphodiesterase Q01065 aa 52-103.XD88347 was identified to be the same as EST R00718 and similar toDrosophila cAMP dependent phosphodiesterase P12252. Both of thesesequences were placed in a category described as having strong homologyto known protein sequences.

A search of the Sequence Tagged Sites (STS) database at NCBI revealedhomology of the 3′-end of PDE10 to STS WI-13322 which has been mapped toregion 220.72 cr. from the top of chromosome 21. The cDNA that this STSwas derived from begins at nucleotide 1899 of FB68.2, does not have thepoly A tail and extends further 3′ than FB68.2. It seems likely thatthis STS sequence represents a PDE10A transcript to which no poly (A⁺)tail has been added or a PDE10A transcript that uses an alternative sitefor poly (A⁺) addition. STS WI-13322 was placed on a Whitehead map ofchromosome 21 near SGC35805, which is derived from the gene for thecystathionine beta-synthase (CBS). CBS has been mapped to chromosome 21at 21q22.3. [Avramopoulos, et al, Hum. Genet. 90:566-568 (1993); Munkeet al., Hum. Genet. 0.42:550-559 (1988)].

A number of different genetic diseases map to this region of chromosome21, for example, Down syndrome [Delabar, et al., Eur. J. Hum. Genet.1:114-124(1993)]. It is not clear that PDE10A falls within the Downsyndrome critical region (DSCR) but it is possible that genes elsewhereon chromosome 21 also contribute to Down syndrome [Korenberg, et al.,Proc. Natl. Acad. Sci. (USA) 91:4997-5001 (1994)]. As another example, alocus involved in bipolar affective disorder in some families has beenmapped to 21 q22.3 [Vallada, et al., J. Affect. Disord. 41:217-221(1996)]. Other examples include Knobloch syndrome, characterized bymyopia and retinal degeneration and detachment [Sertie, et al., Hum.Mol. Genet. 5:843-847 (1996)], and one or more genes responsible forcongenital recessive deafness (DFNB8, DFNB10) [Veske, et al., Hum. Mol.Genet. 5:165168 (1996); Bonne-Tamir, et al., Am. J. Hum. Genet.58:1254-1259 (1996)]. PDE10A may play a role in any or all of thesedisease states.

EXAMPLE 4 Expression and Characterization of PDE10

The entire open reading frame of the PDE10 cDNA (clone FB94a) was placedinto a yeast ADH vector including the alcohol dehydrogenase promoter.The construct was built in two steps.

The 5′end was generated using PCR and FB94a DNA as template. PCR wascarried out using the 5′ primer below (SEQ ID NO: 25) in combinationwith 3′ primer R71A3 (SEQ ID NO: 26). The 5′ primer includes an NcoIsite (underlined in SEQ ID NO: 25 below) and the initiating methioninecodon of FB94a is in bold. The 5′ primer also adds a FLAG® epitope tag(Eastman Kodak, Rochester, N.Y.) to the amino terminus of the encodedprotein; the FLAG® tag is an epitope (SEQ ID NO: 24) recognized by themonoclonal antibody M2 (Eastman Kodak). FLAG ® TAG (SEQ ID NO: 24)Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys 5′Primer (SEQ ID NO: 25)TAGACCATGGACTACAAGGACGACGA- TGACAAGATGGACGCATTCAGAAGCACT R71A3 (SEQ IDNO: 26) CGAGGAGTCAACTTCTTGPCR was carried out using 5 μl each primer (100 μg/ml stock), 5 μl 10×buffer (Perkin Elmer), 5 μl 10× nucleotides (2 mM stock), 3 μl MgCl₂ (25mM stock), FB94a DNA, and 0.3 μl Taq polymerase (Perkin Elmer) in areaction volume of 50 μl. After incubating the reaction mixture at 94°C. for four minutes, 30 cycles of one minute at 94° C., two minutes at50° C., and four minutes at 72° C. were carried out. The PCR product wascleaved with NcoI and HincII and purified using agarose gelelectrophoresis. The 3′sequence of PDE10 was isolated as a HincII/EcoRIfragment cleaved from FB94a and purified by agarose gel electrophoresis.The two fragments were combined and ligated into a NcoI/EcoRI-digestedBluescript® vector (Stratagene, La Jolla, Calif.), previously modifiedby the insertion of the ADH promoter previously removed from aYEpC-PADH2d vector [Price et al. Meth. Enzymol. 185:308-315 (1990)] as aSacI/NcoI fragment, to generate plasmid PDE10-1. New junctions andsequence generated by PCR were verified by sequencing.

In the second step of plasmid construction, the SacI/SalI fragment fromPDE10-1 containing the ADH promoter and PDE10 open reading frame waspurified by two rounds of agarose gel electrophoresis and ligated intoSacI/SalI cut YEpC-PADH2 vector.

Following transformation into BJ2-54, a yeast strain lacking endogenousPDE activity, a colony was selected, streaked out on SC-leu plates and asingle colony carrying the PDE10 construct was chosen for furthercharacterization. Following overnight growth in SC-leu media the culturewas diluted 1:250 in fresh SC-leu and grown overnight at 30° C. until itreached a density of 10⁷ cells/ml. The cells were collected bycentrifugation, washed once with YEP 3% glycerol media, resuspended inYEP containing 3% glycerol, and grown at 30° C. for another 24 hours.The cells were harvested by centrifugation, washed with water, andfrozen at −70° C. until use. Prior to use, an aliquot of the yeastextract was analyzed by SDS PAGE. A protein specific to yeast carryingthe PDE10 expression construct that migrated on the SDS PAGE gels withthe expected mobility (55.5 kDa) was observed by Coomassie bluestaining.

Yeast cells (1×10¹⁰) were thawed with 200 μg/ml each of pepstatin A,leupeptin, and aprotinin 1 mM DTT, and 20 μg/ml calpain inhibitors (Iand II). Two hundred μl of glass beads (0.5 mm, acid washed) were added,and the mixture was vortexed for eight cycles of 30 seconds each.Samples were cooled for 4.5 minutes at 4° C. between cycles. Afterlysis, 0.8 ml lysis buffer was added, the lysate separated from thebeads, and the lysate centrifuged for 30 minutes at 100,000×g in aBeckman TL-100 tabletop centrifuge. The supernatant was aliquoted,frozen in dry ice/ethanol, and stored at −70° C.

Kinetic assays were performed on a BIOMEK® 1000 programmable roboticstation (Beckman Instruments). The range of final substrateconcentration was 0.2 to 1000 μM for cAMP and 0.6-2000 nM for cGMP. Thehighest nucleotide concentration contained 1 to 1.5 million Cerenkovcounts of ³²P-labeled substrate per assay. The enzyme preparation wasinitially diluted 1:500 (cAMP as substrate) or 1:50,000 (cGMP assubstrate). The enzyme dilution buffer consisted of 25 mM Tris-HCl pH8.9, 5 μM ZnSO₄ 5 mM MgCl₂, 1.0 mM DTT, 100 mM NaCl and 0.1 mg/ml BSA(Calbiochem; fatty acid free). Activity at each substrate concentrationwas derived from a linear fit of successive four-fold enzyme dilutionsacross the plate.

Assays were performed at 30° C. for 15 minutes. After 12 minutes, 5 μlsnake venom from Crotalus atrox (15, mg/ml protein) was added to eachreaction. Assays were stopped by addition 200 μl of charcoal suspension(25 mg/ml activated charcoal in 0.1 M monobasic potassium phosphate).The plate was centrifuged at 2600 rpm, and 200 μl of each supernatantwas transferred into Microbeta® counting plates and counted on a WALLACMicrobeta® by Cerenkov counting. Data were evaluated with a predesignedMicrosoft Excel® Spreadsheet, and the kinetic parameters were fitted toa Michaelis-Menton model using the program Table Curve® from JandelScientific.

Results indicated that the K_(m) for cGMP hydrolysis was 5 (±1) nM andthe K_(m) for cAMP hydrolysis was 160 (±30) μM. In the extract, cGMPhydrolytic activity was determined to be 0.035 (±0.01) μmol/min/mg,while cAMP hydrolysis was measured to be 0.52 (±0.06) μmol/min/mg. Thus,although PDE10 had much greater affinity for cGMP, the V^(max) for cAMPwas 15-fold greater.

In order to distinguish PDE10 from other PDE families, a panel of PDEinhibitors with activities against defined PDE families was tested forPDE10 inhibition using cAMP as a substrate. The results of the assay areset out in Table 1 below. TABLE 1 PDE10 Inhibition with Isozyme-specificPDE Inhibitors Target PDE10 Target Family Inhibitor Family IC₅₀ (μM)IC₅₀ (μM) SCH46642 PDE1 14 0.2⁵ EHNA PDE2 477 0.8² Cilostamide PDE3 1000.04-0.9³ Rolipram PDE4 529 0.18-0.5⁴ DMPPO PDE5 9 0.003¹ IBMXnon-specific 59   2-20¹¹Coste and Grodin, Biochem. Pharmacol. 50:1577-1585 (1995).²Podzuweit, et al. Cell. Signaling 7:733-738 (1995)³Manganiello et al., in Isoenzymes of Cyclic NucleotidePhosphodiesterases, Beavo and Houslay (Eds.), John Wiley and Sons, Ltd., pp. 87-116 (1990)⁴Bolger et al., Mol. Cell. Biol. 13:6558-6571 (1993)⁵Ahn, et al., Abstract from the 9th International Conference on SecondMessengers and Phosphoproteins, Nashville, TN, 1995, p. 86.

The results further distinguish PDE10 from PDEs in families 1 through inthat specific inhibitors for enzymes in those families are significantlyless effective in inhibiting PDE10.

EXAMPLE 5 Production of Anti-PDE10 Antibodies

A GST fusion protein was produced in E. coli to provide an antigen forgeneration of monoclonal antibodies to PDE10. An EcoRI fragment fromFB76.2 (nucleotides 280 through 1829 in SEQ ID NO: 18) was inserted intothe EcoRI site of pGEX3X (Pharmacia) and the resultant construct wastransformed in the E. coli strain XL1 Blue. A GST-PDE10 fusion proteinincluding 464 amino acids from PDE10 was expressed from this constructfollowing induction with IPTG. The fusion protein was isolated usingSDS-PAGE, the band of appropriate size excised from the gel followingstaining with cold 0.4 M KCl, and the protein obtained from theacrylamide by electroelution. The elution product was dialyzed againstPBS and concentrated using Centriprep 10 and Centricon columns (Amicon,Beverly Mass.) prior to being injected into mice.

On day 0, four Balb/c mice were pre-bled and immunized by subcutaneousinjection with a panel of antigens including 30 μg/mouse GST-PDE10fusion protein in complete Freund's adjuvant in 200 μl total volume. Thesame injections were repeated at weeks three and nine in incompleteFreund's adjuvant. Ten days after the last immunization, test bleedswere obtained and screened by antigen capture ELISA and Westernanalysis.

In the ELISA, Immulon® 4 plates (Dynex, Cambridge, Mass.) were coated at4° C. with 50 μl/well of a solution containing 2 μg/ml GST-PDE10 in 50mM carbonate buffer, pH 9.6. Plates were blocked with 0.5% fish skingelatin (Sigma) for 30 minutes and 50 μl serum diluted in PBS with 0.5%Tween® 20 (PBST) was added. Serum dilutions ranged from 1:100 to1:102,400 and were obtained by a series of doubling dilutions. Afterincubation at 37° C. for 30 minutes and washing three times with PBST,5.0 μl of horseradish peroxidase-conjugated goat anti-mouse IgG(fc)antibody. (Jackson) (diluted 1:10000 in PBST) was added. Plates wereincubated as above and washed four times with PBST. Antibody wasdetected with addition of tetramethyl benzidine (Sigma Chemical, St.Louis, Mo.) and the color reaction was stopped after five minutes withthe addition of 50 μl of 15% H₂SO₄. Absorbance at 0.450 nM was measuredon a plate reader.

For Western analysis, SDS-PAGE gels were run with approximately 10 μgyeast PDE10 extract and approximately 200 ng of gel-purified GST-PDE10and the proteins were transferred to Immobilon-PVDF. A standard enhancedchemiluminescence (ECL) Western blot protocol was performed using BioRadgoat anti-mouse IgG horseradish peroxidase as the secondary antibody.

In preparation of hybridomas, splenocytes from mice giving a positiveresult from the ELISA and/or Western blotting protocols above, werefused to NS-1 cells in a ratio of 5:1 by standard methods usingpolyethylene glycol 1500 (Boehringer Mannheim) [Harlow and Lane,Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, p.211(1988)]. The fused cells were resuspended in 200 ml RPMI containing 15%FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine(HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ murinethymocytes/ml and dispensed into ten 96-well flat bottom tissue cultureplates (Corning, United Kingdom) at 200 μl/well. Cells were fed on days2, 4, and 6 post fusion by aspirating approximately 100 μl from eachwell with an 18 G needle (Becton Dickinson) and adding 100 μl/wellplating medium described above except containing 10 units/ml IL-6 andlacking thymocytes. On days 9 to 12, supernatants from the fusion wellswere screened by antigen capture ELISA using GST and GST-PDE10 and byECL Western analysis as described above.

A positive signal of the expected size was obtained on both lanes of theWestern blot using mouse blood and monoclonal antibodies with reactivityto the yeast recombinant protein were obtained in the subsequent fusion.

EXAMPLE 6 Analysis of PDE10 Expression by In Situ Hybridization

Expression of PDE10 was examined in tissue sections by in situhybridization as described below.

Preparation of Probe

An EcoRI/PstI restriction enzyme fragment from the cDNA FB93a(corresponding to nucleotides 370 through 978 in SEQ ID NO: 22) wassubcloned into a Bluescript® vector (Stratagene, La Jolla, Calif.) togenerate an expression plasmid designated PDE10A3A. The plasmid wasdigested with EcoRI and transcribed with T3 polymerase to generate anantisense probe. A sense probe was generated by digestion the plasmidwith BamHI and transcribing with T7 polymerase. The PDE10 templates weretranscribed using a RNA Transcription kit (Stratagene, La Jolla, Calif.)in a reaction containing 5 μl of 5× transcription buffer (Stratagene),30 mM DTT (Stratagene), 0.8 mM each ATP, CTP, GTP (10 mM (Stratagene),40 U RNase Block II (Stratagene), 12.5 U T3 or T7 polymerase(Stratagene), and 300 ng linearized plasmid template, 50 μCi ³⁵S-UTP(greater than 1000 Ci/mmol, Amersham, Arlington Heights, Ill.). Themixture was incubated at 37° C. for one hour after which the templateDNA was removed by addition of 1 μl of RNase-free DNase I (Stratagene)and incubation for 15 minutes at 37° C. The probe was hydrolyzed toapproximately 250 nucleotides in length to facilitate tissue penetrationby adding 4 μl 1 M NaHCO₃ and 6 μl 1 M Na₂CO₃ for 22 minutes at 60° C.and the reaction mixture was neutralized by addition of 25 μl of asolution containing 100 μl 3 M sodium acetate, 5 μl acetic acid (VWR,So. Plainfield, N.J.), and 395 μl dH₂O. A Quick Spin G50 RNA column(5′→3′ Inc., Boulder, Colo.) was prepared according to themanufacturer's suggested protocol. The probe was placed in the center ofthe column and the column centrifuged for four minutes at 1,000 rpm in adesk top centrifuge. The column flow-through was mixed with 50 μl dH₂O,2 μl of a 10 mg/ml tRNA solution, 10 μl 3 M sodium acetate, and 200 μl100%, ethanol (VWR) and the resulting mixture was incubated at −20° C.overnight. The probe solution was microfuged for 15 minutes at 4° C.,the supernatant was removed, and the pellet was resuspended in 40 μl1×TBE containing 1 μl of 0.1 M DTT. The probe was stored at −70° C.until the in situ hybridization assay was performed.

Preparation of Tissue Samples and In Situ Hybridization

Tissues (National Disease Research Interchange, Philadelphia, Pa. andCooperative Human Tissue Network, Philadelphia, Pa.) were sectioned at 6μm and placed on Superfrost Plus slides (VWR). Sections were fixed for20 minutes at, 4° C. in 4% paraformaldehyde (Sigma, St. Louis, Mo.). Theslides were rinsed in three changes of 1× calcium-, magnesium-freephosphate buffered saline (CMF-PBS), dehydrated with three successivewashes with 70% ethanol, 95% ethanol and 100% ethanol, and dried, for 30minutes at room temperature. The slides were placed in 70% formamide(J.T. Baker) in 2×SSC for two minutes at 70° C., rinsed in 2×SSC at 4°C., dehydrated through 70%, 95% and 100% ethanol washes, and dried for30 minutes at room temperature.

A prehybridization step was performed by placing the slides in anairtight box containing a piece of filter paper saturated with buffercontaining 50% formamide (J.T. Baker) in 4×SSC. Each section was coveredwith 100 μl of rHB2 buffer consisting of 10% dextran sulfate (Sigma),50% formamide (J.T. Baker, Phillpsburg, N.J.), 100 mM DTT (BoehringerMannheim, Indianapolis, Ind.), 0.3 M NaCl (Sigma), 20 mM Tris, pH 7.5, 5mM EDTA (Sigma), and 1× Denhardt's solution (Sigma) and the slides wereincubated at 42° C. for two hours. The probe, as described above, wasprepared by mixing 4×10′ cpm/tissue section with 5 μl of a 10 mg/ml tRNAsolution per section and heating the mixture at 95° C. for threeminutes. Ice cold rHB2 buffer was added to bring the final volume to 20μl/section. The probe-containing solution (20 μl/section) was added to100 μl rHB2 buffer previously applied. The slides were incubated at 55°C. for 12 to 16 hours. Following hybridization, the slides were washedonce in 4×SSC containing 10 mM DTT for one hour at room temperature;once in 50% deionized formamide (J.T. Baker), 1×SSC, and 1 mM DTT for 40minutes at 60° C. once in 2×SSC for 30 minutes at room temperature, andonce in 0.1×SSC for 30 minutes at room temperature. The sections weredehydrated through 70%, 95%, and 100% ethanol washes and air dried for30 minutes. The slides were dipped in Kodak NTB2 nuclear emulsion, driedfor one to three hours at room temperature in the dark, and stored inthe dark at 4° C. with desiccant until time of development. The slideswere developed in 4° C. Kodak Dektol® developer for two minutes, dippedfour times in 4° C. dH₂O, and placed in 4° C. Kodak fixer for tenminutes. The slides were rinsed in dH₂O and a standard hematoxylin andeosin (H&E) stain was performed as follows.

The slides were rinsed in dH₂O and stained with hematoxylin and eosin bytransfer of the slides through a series of the following steps: fiveminutes in formaldehyde/alcohol (100 ml formaldehyde, 900 ml 80%ethanol); three rinses in water for a total of two minutes; five minutesin 0.75% Harris hematoxylin (Sigma); three rinses in water for a totalof two minutes; one dip in 1% HCl/50% ethanol; one rinse in water; fourdips in 1% lithium carbonate; ten minutes in tap water; two minutes in0.5%, eosin (Sigma); three rinses in water for a total of two minutes;two minutes in 70% ethanol; three one-minute rinses in 95% ethanol; twoone-minute rinses in 00% ethanol; and two two-minute rinses in xylene.Slides were mounted with cytoseal 60 (Stephens Scientific, Riverdale,N.J.).

The signals obtained with an antisense PDE10 probe were compared to thecontrol signals generated by a sense PDE10 probe and any signalspecific, to the antisense probe was assumed to represent PDE10expression. PDE10 signal was detected throughout much of the cerebellum,with very strong signal in the Purkinje cells.

EXAMPLE 7 High Throughput Screening for PDE10 Inhibitors

In an attempt to identify specific inhibitors, PDE10 was screenedagainst a chemical library containing compounds of known structure.Initial screening was performed on pools of compounds (22 compounds perpool) with each compound present at 4.6 μM. Pools which inhibited PDE10activity by greater than 50% were selected and the individual compoundsin the pool were screened at a concentration of 20 μM. IC₅₀ values weredetermined for compounds that inhibited enzyme activity.

An extract was prepared from Saccharomyces cerevisiae strain BJ2-54(described in Example 4) lacking endogenous PDE activity and havingPDE10 at an activity of 49 mmol cGMP hydrolyzed/min/ml with 32 nM cGMP.The extract was diluted 1:21,000-fold for use in the assay. Dilutionbuffer included 25 mM Tris, pH 8.0, 0.1 mM DTT, 5.0 mM MgCl₂, 100 mMNaCl, 5 μM ZnSO₄ and 100 μg/ml BSA. PDE assay buffer (5×) contained 200mM Tris, pH 8.0, 5 mM EGTA, 25 mM MgCl₂ and 0.5 mg/ml BSA. Just prior toscreening. 5×PDE assay buffer, deionized water, and 5′-nucleosidase(stock solution 15 mg/ml snake venom 5′-nucleosidase in 20 mM Tris, pH8.0) were mixed at ratios of 4:4:1 to make Assay Reagent Mix.

A Packard MultiPROBE® was used to add 45 μl of the Assay Reagent Mix and20 μl of the chemical compound pools. A BIOMEK® 1000 (See Example 4) wasused to add 20 μl of PDE10 extract diluted as described above and 20 μl³²P-cGMP (ICN, specific activity 250 μCi/mmol, diluted to 0.4 μCi/ml, 16nM, in deionized water). Final cGMP concentration in the assay was 0.08μCi/ml, 3.2 nM. Ten minutes after addition of ³²P-cGMP, 140 μl of 25mg/ml charcoal (in 0.1 M NaH₂PO₄) was added to stop the reaction. Aftera 20 minute incubation at room temperature, the assay plates werecentrifuged for five minutes at 3,500 rpm in a Beckman GS-6R centrifuge.A BIOMEK® 1000 was used to transfer 140 μl of the supernatant to aWallac counting plate and Cerenkov radiation was measured in a WallacMicroBeta Counter.

Several compounds that merit further investigation were found to inhibitenzyme activity.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

1. A purified and isolated PDE10 polypeptide. 2.-33. (Canceled)